Mesoporous calcium silicate compositions and methods for synthesis of mesoporous calcium silicate for controlled release of bioactive agents

ABSTRACT

Mesoporous calcium silicate compositions for controlled release of bioactive agents and methods for producing such compositions are disclosed herein. In one embodiment, mesoporous calcium silicate is synthesized by acid modification of wollastonite particles using hydrochloric acid. A hydrated silica gel layer having abundant Si—OH functional groups can be formed on the surface of wollastonite after acid modification. Bruhauer-Emmett-Teller (BET) surface area increased significantly due to acid modification and, in one arrangement, reached over 350 m 2 /g. Acid modified mesoporous calcium silicate compositions show a higher ability to adsorb protein compared to unmodified particles and demonstrate controlled release kinetics of these proteins.

CROSS-REFERENCE TO APPLICATION(S) INCORPORATED BY REFERENCE

The present application claims priority to U.S. Provisional PatentApplication No. 60/972,619 filed Sep. 14, 2007, entitled “METHODS FORSYNTHESIS OF MESOPOROUS CALCIUM SILICATE FOR CONTROLLED RELEASE OFPROTEIN,” and incorporated herein in its entirety by reference. Thepresent application incorporates the subject matter of InternationalPublication No. WO/2007/124511, entitled “RESORBABLE CERAMICS WITHCONTROLLED STRENGTH LOSS RATES,” filed Apr. 25, 2007, and U.S.Publication No. 2007/0203584 A1, entitled “BONE REPLACEMENT MATERIALS,”filed Feb. 14, 2007, in their entirety by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This work was partially funded by the National Science Foundation (Grant#0134476) and the Office of Naval Research (Grant Nos: N00014-1-04-0644and N00014-1-05-0583), and the United States government has, therefore,certain rights to the present invention.

TECHNICAL FIELD

The present disclosure is generally directed to mesoporous calciumsilicate compositions for use in bone regeneration and other biomedicalapplications, and directed to synthesis of mesoporous calcium silicatefor controlled release of bioactive agents, such as proteins and/orpharmaceutical agents.

BACKGROUND

Bone forming growth factors, such as bone morphogenetic protein (BMP)and transforming growth factor (TGF-β) have been widely investigated inorthopedics and tissue engineering with great success. These moleculesstimulate bone cell growth and differentiation, and accelerate bonetissue regeneration [1, 2]. To prevent possible side-effects innon-target tissues, delivery of growth factors are performed in acontrolled manner. For example, growth factors can be sequentiallyadministered in a manner that mimics the time profile of the healingprocess [2-4].

Biomaterials, such as biopolymers [5-8], inorganic ceramics [9-18] andtheir compositions [19, 20], can act as reservoirs for deliverableprotein and other bioactive molecules if they demonstrate a highcapacity for protein adsorption while preserving the protein structureand biological activity over time. In addition to controlled proteindelivery, carriers that are biocompatible with bone tissue can alsoserve as a scaffold for new tissue formation during normal tissue repairor healing. Furthermore, such biomaterials should be non-immunogenicand, in many cases, biodegradable once tissue regeneration is complete[2, 3]. Typically, biopolymers are not bioactive because they do notchemically bond to living bone. Inorganic bone replacement materials,including calcium phosphate ceramics [10, 11], bioglass andbioglass-ceramics [12], silica gel [13,14], and calcium phosphatecements [15-18], have been extensively investigated due to theirexcellent bioactivity with bone tissue. However, the adsorption capacityof protein on these carriers is limited to outer surface adsorption.Moreover, protein release from these materials displays a burst release(e.g., rapid release) due to the weak bonding between protein and thecarriers.

BRIEF DESCRIPTION OF THE DRAWINGS

In order that advantages of the disclosure will be readily understood, amore particular description of aspects of the disclosure brieflydescribed above will be rendered by reference to specific embodimentsand the appended drawings. Understanding that these drawings depict onlytypical embodiments of the disclosure and are not therefore to beconsidered to be limiting of its scope, the disclosure will be describedand explained with additional specificity and detail through the use ofthe accompanying drawings.

FIGS. 1A-1C illustrate a reaction mechanism of (A) wollastonitechain-silicate structure consisting of a network of covalently bondedsilica that is interrupted and modified by Ca cations, (B) a reactionstep wherein weakly bonded, network-modifying Ca is released relativelyquickly to solution as Ca is exchanged for hydrogen ions and (C) areaction step wherein silanol (Si—OH) in the leach layer repolymerizesto form a silica gel, and in accordance with an embodiment of thedisclosure.

FIG. 2 is a XRD spectra of calcium silicate before and after acidmodification in accordance with an embodiment of the disclosure.

FIG. 3 is a FTIR spectra of calcium silicate before and after acidmodification in accordance with an embodiment of the disclosure.

FIG. 4 is a ²⁹Si magic-angle spinning (MAS) nuclear magnetic resonance(NMR) analysis of calcium silicate before (CS) and after (CS-7) acidmodification in accordance with an embodiment of the disclosure.

FIGS. 5A-5C are, respectively, SEM images of morphologies andmicrostructures of calcium silicate after acid treatment at (A) pH 7.0,(B) pH 4.5 and (C) pH 0.5, and in accordance with an embodiment of thedisclosure.

FIGS. 6A-6C are, respectively, graphical representations of a sizedistribution of (A) CS-7 mesoporous calcium silicate, (B) CS-4.5mesoporous calcium silicate and (C) CS-0.5 mesoporous calcium silicatein accordance with an embodiment of the disclosure.

FIGS. 7A-7B are, respectively, graphical representations of the kineticsof BSA adsorption on calcium silicate and mesoporous calcium silicate at(A) pH 7 and (B) pH 4.5 in accordance with an embodiment of thedisclosure.

FIG. 8 is a graphical representation of the kinetics of lysozymeadsorption on calcium silicate and mesoporous calcium silicate inaccordance with an embodiment of the disclosure.

FIG. 9 is a FTIR spectra of mesoporous calcium silicate before andfollowing lysozyme adsorption in accordance with an embodiment of thedisclosure.

FIG. 10 is a graphical representation of nitrogen adsorption isothermsof mesoporous calcium silicate before and following lysozyme adsorptionin accordance with an embodiment of the disclosure.

FIGS. 11A-11B are, respectively, graphical representations of cumulativerelease kinetics of lysozyme from calcium silicate at (A) pH 7 and (B)pH 4.5 in accordance with an embodiment of the disclosure.

FIG. 12 is a graphical representation of a degradation rate ofmesoporous calcium silicate at pH 7 and pH 4.5 in accordance with anembodiment of the disclosure.

FIG. 13 is a graphical representation of cell densities of human fetalosteoblast cells cultured with samples of calcium silicate andmesoporous calcium silicate in accordance with an embodiment of thedisclosure.

FIGS. 14A-14B are SEM images of morphologies of cells cultured onpressed mesoporous calcium silicate after 3 days (14A) and 7 days (14B)of culture in accordance with an embodiment of the disclosure.

DETAILED DESCRIPTION

The present disclosure describes mesoporous calcium silicatecompositions and methods for producing mesoporous calcium silicate forcontrolled release of bioactive agents. In some embodiments, bioactiveagents can include proteins, growth factors (e.g., osteoinductive growthfactors), pharmaceutical drugs, antibiotics, antimicrobial agents,polypeptides, etc. In one embodiment, mesoporous calcium silicate issynthesized by acid modification of wollastonite (e.g., calciumsilicate) particles by adding hydrochloric acid to a wollastonite slurryuntil a neutral (e.g., 7.0) or acidic (less than 7.0) pH level isachieved. A hydrated silica gel layer having abundant Si—OH functionalgroups can be formed on the surface of calcium silicate particlesfollowing acid modification. In further embodiments, acid modificationof calcium silicate particles increases particle surface area, asmeasured by the Bruhauer-Emmett-Teller (BET) method. Acid modifiedmesoporous calcium silicate particles demonstrate an increased abilityto adsorb protein compared to unmodified calcium silicate particles, andfurther demonstrate controlled release kinetics of these proteins.Accordingly, the compositions disclosed herein provide calcium silicatematerials for use in bone implant applications having increasedbioactivity and bioactive agent loading.

It will be appreciated that several of the details set forth below areprovided to describe the following embodiments in a manner sufficient toenable a person skilled in the relevant art to make and use thedisclosed embodiments. Several of the details and advantages describedbelow, however, may not be necessary to practice certain embodiments ofthe disclosure. Additionally, the disclosure can include otherembodiments that are within the scope of the claims but are notdescribed in detail with respect to FIGS. 1A-14B.

A. Embodiments of Mesoporous Calcium Silicate Compositions and Methodsfor Preparing and Using such Compositions

Particular aspects of the present disclosure provide mesoporous calciumsilicate compositions for controlled release of bioactive agents to beused for or used with bone replacement materials (e.g.,three-dimensional tissue scaffolds). Mesoporous calcium silicatecompositions can be used alone or in combination with otherbiocompatible scaffold materials (e.g., calcium phosphates [CaP] with orwithout dopants to facilitate controlled strength loss rates), such asthose described in International Publication No. WO/2007/124511,incorporated herein by reference. Porous, interconnected scaffolds canallow for and facilitate tissue (e.g., bone) cells to grow andproliferate such that the developing tissue(s) can replace the temporaryscaffold material. For example, the scaffold material can have acontrolled strength loss. In other embodiments, the mesoporous calciumcomposition can be a coating material.

In one embodiment, the mesoporous calcium silicate composition isbiocompatible with bone or other tissue in the body of a human or otheranimal. For example, the compositions can be used alone or incombination with additional materials for dental implants, orthopedicimplants, craniomaxillofacial applications, spinal grafting, etc. Inother embodiments, the mesoporous calcium silicate compositions can beused alone, as a complement, and/or as an addition to other materialsfor forming bone implants and/or coatings for bone implants. In furtherembodiments, mesoporous calcium silicate compositions as disclosedherein can be packed into bone graft material (e.g., hydroxylapatite,bioactive glass, calcium phosphate ceramics, etc.), or used in bonepastes for providing a bioresorbable and biocompatible composition forregeneration or restoration of musculoskeletal tissue. In anotherembodiment, the mesoporous calcium silicate compositions can providecoatings for other types of bone grafts, such as an allograft orxenograft.

In some embodiments, the composition promotes bone in-growth and/orrepair of damaged tissue. In vitro and in vivo studies have shown thatcalcium silicate ceramics could induce a bone-like apatite layerformation in simulated body fluid (SBF) [27] and chemically integrateinto the structure of living bone tissue [28]. In one embodiment, thecomposition is used for regenerating tissue. The tissue can include atleast one of bone, cartilage, muscle and musculoskeletal tissue. In manyembodiments disclosed herein, the mesoporous calcium silicate isbiocompatible as well as bioresorbable (e.g., allow the native tissuesto gradually replace the implanted material). In one embodiment, themesoporous calcium silicate is biocompatible with respect to at leastone of eukaryotic cells, mammalian cells, bone forming cells, osteoblastcells, cartilage cells, muscle cells, stem cells, differentiated stemcells, bone marrow stem cells and nerve cells.

Some embodiments of mesoporous calcium silicate compositions furtherinclude at least one bioactive agent. Bioactive agents can includeproteins, polypeptides, growth factors (e.g., human growth factors,osteoinductive growth factors), morphogens (e.g., bone morphogenicproteins), pharmaceutical drugs (e.g., a disease-specific drug, anosteoporotic drug, a symptom-treating drug, a pain relieving drug,etc.), chemicals, antibiotics, antimicrobial agents, vitamins (e.g.,vitamin D, etc.), etc. The bioactive agent can be deposited on asurface, or in another embodiment, incorporated into the mesoporouscalcium silicate compositions. In further embodiments, the bioactiveagent can be stored within and/or on a surface of a scaffold material.

In many of the preceding embodiments, the bioactive agent can beincorporated or stored within the composition and/or scaffold materialto provide for release and, more particularly, controlled release of theagent to facilitate restoration or regeneration of bone or other tissue.In some embodiments, the bioactive agent can be selectively releasedfrom the composition using one or more triggering mechanisms. Forexample, a triggering mechanism, such as application of electrical,magnetic, chemical or photochemical triggers can be used to selectivelyrelease the bioactive agent from the mesoporous calcium silicatecomposition at a time following implant. In some instances, thetriggering event can include chemical ingestion, injection or infusion;exposure to UV light, ultrasound, magnetic fields, electric current,etc.

Wollastonite (e.g., calcium silicate) is a chain-silicate mineral whichconsists of a network of covalently bonded silica that is interruptedand modified by Ca²⁺ cations [33, 34]. In one embodiment, synthesis ofcalcium silicate can include preparing a precipitation reaction ofCa(NO₃)₂.4H₂O and Na₂SiO₃.9H₂O at a 1:1 ratio.

FIGS. 1A-1C illustrate a reaction mechanism of (A) wollastonitechain-silicate structure consisting of a network of covalently bondedsilica that is interrupted and modified by Ca cations, (B) a reactionstep wherein weakly bonded, network-modifying Ca is released relativelyquickly to solution as Ca is exchanged for hydrogen ions and (C) areaction step wherein silanol (Si—OH) in the leach layer repolymerizesto form a silica gel, and in accordance with an embodiment of thedisclosure. Referring to FIGS. 1A and 1B, the weakly bonded,network-modifying Ca²⁺ cations are released to solution (e.g., SBF) asthey are exchanged for hydrogen ions, resulting in the formation ofSi—OH, as shown below:

≡—Si—O—Ca—O—Si≡+→2≡Si—OH+Ca²⁺

Silanol (Si—OH) can further repolymerize (FIG. 1C) to form silica gel bythe following reaction:

≡Si—OH+HO—Si≡→≡Si—O—Si≡+H₂O.

In one embodiment, surface modification of wollastonite is applied byacid treatment, for example, to accelerate Si—OH formation onwollastonite particles and to form a mesoporous structure on the surfaceof wollastonite. Following acid modification, Ca²⁺ is quickly leachedfrom the wollastonite particles leaving only low or no Ca in an upperlayer of the wollastonite particles. For example, calcium silicateformed from the precipitate reaction of Ca(NO₃)₂.4H₂O and Na₂SiO₃.9H₂O,can be acid modified using a hydrochloric acid (HCl) solution. In someembodiments, HCL, or other acid, can be added to a slurry of calciumsilicate until a neutral (e.g., 7.0) or acidic (less than 7.0) pH levelis achieved. In further embodiments, HCL, or other acid, can becontinuously added to maintain a selected pH (e.g., approximately pH7.0, less than pH 7.0, less than pH 5.0, less than pH 1.0, approximatelypH 0.5, etc.) for modification of the calcium silicate particles.

In some embodiments, acid modified calcium silicate particles have amesoporous surface with nanoparticles. In some embodiments, the size ofthe nanoparticles is dependent on the conditions of the acid treatment(e.g., pH conditions, length of treatment, etc.). For example, in oneembodiment, the size of the nanoparticles can be between 20 and 60 nm.In other embodiments, the size of the nanoparticles can be between 20and 30 nm, between 40 and 60 nm, less than 75 nm, less than 30 nm, etc.The mesoporous surface of the acid-modified calcium silicate particlescan have, in one embodiment, an average pore diameter of approximately 5nm to approximately 4 nm. In other embodiments, the average porediameter can be greater than 10 nm, less than 4 nm, between 10 nm and 4nm, etc. The average pore diameter of the mesoporous structure candepend on the conditions of the acid treatment (e.g., pH conditions,length of treatment, etc.). Accordingly, one of ordinary skill in theart will recognize that the mesoporous calcium silicate particles can beformulated (e.g., using variable acid-modification conditions) such thatthe nanoparticle size and the average pore diameter are relativelyselectable.

Acid modification of calcium silicate particles can also significantlyincrease a surface area of the particles (e.g., through formation of themesoporous structure). Accordingly, mesoporous calcium silicate providesan increased surface area for adsorbing protein molecules and/or otherbioactive materials. As such mesoporous calcium silicate shows higherprotein adsorption, for example, than non-acid treated calcium silicate.

In some embodiments, it is desirable to have bioactive agents releasedcontinually over a certain or selectable period of time during a healingprocess or other tissue generation process. For unmodified calciumsilicate, bioactive agents (e.g., protein) are adsorbed on the materialsurface by weak physical forces, e.g., Van Der Waals and electrostaticinteraction [35]. Due to the nature of these bonding forces, thebioactive agents adsorbed by unmodified calcium silicate release quicklyin the first several hours. In contrast, mesoporous calcium silicatecompositions (e.g., acid-modified calcium silicate particles) haveabundant O—H and Si—OH functional groups exposed on particle surfaces.Accordingly, bioactive agents (e.g., protein) can be bound to themesoporous particle surface via hydrogen bonding with the Si—OH groups.Hydrogen bonding is stronger than the weak physical interactionsobserved on unmodified particles. Thus, the release kinetics of adsorbedbioactive agents from mesoporous calcium silicate compositions can berelatively slow and can be controllable.

In one embodiment, a release profile of the bioactive agent from amesoporous calcium silicate composition includes a burst phase and anextended release phase. The burst phase can include an immediate releaseof a portion of the bioactive agent from the composition followed by theextended release phase. The extended release phase can include a slowrelease of the bioactive agent that extends for a plurality of days.

In another embodiment, the disclosure is directed to biocompatiblecompositions for controlled release of bioactive agents in bonereplacement and/or tissue regeneration applications. The biocompatiblecomposition can include mesoporous calcium silicate (CaSi) particles. Insome embodiments, the mesoporous calcium silicate particles can have oneor more bioactive agents adsorbed thereon or adsorbed therein. Forexample, the composition can include mesoporous CaSi particles with acombination of adsorbed bioactive agents, such as a combination ofosteoinductive growth factor and vitamin D.

In a further embodiment, the composition can include a combination ofmesoporous CaSi particles having different characteristics and/orbioactive agents. For example, a composition can include first andsecond portions of mesoporous CaSi particles. The first portion ofmesoporous CaSi particles can have a first nanoparticle size, a firstaverage pore diameter and a first bioactive agent adsorbed thereon fordefining a first release kinetics profile of the first bioactive agent.The second portion of mesoporous CaSi particles can have a secondnanoparticle size, a second average pore diameter and a second bioactiveagent adsorbed thereon for defining a second release kinetics profile ofthe second bioactive agent. One of ordinary skill in the art willrecognize that any number of combinations of mesoporous CaSi particlescan be used to formulate a biocompatible composition having one or moredesired bioactive agents, a desirable release kinetics profile, desiredbioresorbable characteristics (e.g., degradation characteristics), etc.

In a further embodiment, a biocompatible composition for controlledrelease of bioactive agents in bone replacement and/or tissueregeneration applications can include various combinations of mesoporousCaSi particles and other resorbable ceramics, such as ceramicscontaining calcium phosphates (CaP) and calcium sulfates (CaS). In oneembodiment, compositions including CaP and CaSi can be useful forproviding compositions having selectable controlled release profiles forbioactive agents, selectable controlled strength loss rates within aselect period of time, etc.

In some embodiments, a biocompatible composition for controlled releaseof bioactive agents in bone replacement and/or tissue regenerationapplications can include a mesoporous calcium silicate-based materialand/or a Ca-based ceramic (e.g., CaP, CaS) having at least one dopantincluded therein. In one embodiment, the dopant can include metal saltswith metal ions; and in another embodiment, the dopant can include metaloxides. For example, the dopant can include one or more of Zn²⁺, Mg²⁺,Si²⁺, Na⁺, K⁺, Sr²⁺, Cu²⁺, Fe³⁺/Fe²⁺, Ag⁺, Ti⁴⁺, CO₃ ²⁻, F⁻, MgO, ZnO,NaF, KF, FeO/Fe₂O₃, SrO, CuO, Sio₂, TiO₂, Ag₂O and CaCO₃, present in asingle-element or multi-element amount between 0 and about 10 wt %. Inother embodiments, the dopant can be present in an amount from about 0.5to about 5 wt %, from about 1 to about 3 wt %, from about 2 to about wt.%, from about 3 to about 7 wt %, from about 4 to about 7 wt %.

In one embodiment, the dopant is present in an amount sufficient tomaintain the compressive strength of the material (e.g., scaffoldmaterial, coating material, biocompatible composition, etc.) at about30% of original or higher. In other embodiments, the dopant can beincluded in an amount sufficient to maintain the compressive strength ofthe material at about 40% of original or higher, 50% of original orhigher, 60% of original or higher, etc. In other embodiments, the dopantis present in an amount sufficient to maintain the compressive strengthof the material at about 70% to about 90% of original or higher. In afurther embodiment, the dopant is present in an amount sufficient tolower the compressive strength of the material to about 90% of theoriginal strength (e.g., without dopant) or less. In each of the abovedescribed embodiments, the material and/or composition can have acompressive strength maintained for a period of about 3 months to about6 months under body, body fluid or simulated body fluid conditions, toprovide for a fast-degrading composition. In other embodiments, thecompressive strength can be maintained for a period of less than 3months or greater than 6 months.

Accordingly, it can be appreciated that the mesoporous calcium silicateprovides a drug delivery platform for controlled and/or sustainedrelease of bioactive agents, such as protein and/or drugs, for therapyto a subject. For example, when placed in a subject as a graft, acoating, or other composition, the agent-loaded mesoporous calciumsilicate can release the agent for an extended period of time, e.g., aperiod of days or weeks, providing a therapeutic amount of the bioactiveagent to the subject, and at the target site (e.g., for tissue growth,bone replacement, bone regeneration, etc.).

In one embodiment, mesoporous calcium silicate compositions can be usedas a vehicle for protein delivery with controlled release kinetics invivo. Mesoporous calcium silicate can be synthesized from acid treatment(e.g., treatment with hydrochloric acid) of calcium silicate particles(e.g., powder). In one embodiment, mesoporosity of the surface ofcalcium silicate can provide increased surface area for protein (orother bioactive molecule) adsorption and retention as compared tounmodified calcium silicate. Acid treatment of calcium silicate alsopromotes the formation of Si—OH functional groups on the materialsurface, improving both biocompatibility of the particles as well aspromoting strengthened bioactive agent interactions with the materialsurface.

B. Examples of Methods for Synthesis and Characterization of MesoporousCalcium Silicate

The following examples are intended to demonstrate aspects of thedisclosure more fully without acting as a limitation upon the scope ofthe disclosure, as numerous modifications and variations will beapparent to those skilled in the relevant art.

EXAMPLE 1 Preparation of Calcium Silicate

To synthesize calcium silicate, CS powder was prepared from aprecipitation reaction of Ca(NO₃)₂.4H₂O and Na₂SiO₃.9H₂O [31]. In thisexample, 1000 ml of 0.4 mol Ca(NO₃)₂.4H₂O solution was stirred at roomtemperature, while 1000 ml of 0.4 mol Na₂SiO₃.9H₂O was added dropwiseover approximately 40-60 min to produce a white precipitate. The whiteprecipitate was stirred for approximately 4 hours followed by washingfive times with deionized water to remove excess Na⁺ and NO₃ ⁻ ions. Theprecipitate was then washed two times with 100% ethanol to improvedispersion characteristics. The obtained powder was dried atapproximately 80° C. for about 24 hours.

EXAMPLE 2 Preparation of Mesoporous Calcium Silicate

A mesoporous structure of calcium silicate can be produced by acidtreatment of the CS powder prepared as in Example 1 using a hydrochloricacid (HCl) solution. In this example, CS powder was dispersed indeionized water to produce a slurry with a powder/water weight ratio of1:10. The pH of the starting slurry was about 11. Next, the slurry wasseparated into four samples, in which 1N HCl solution was added dropwiseto adjust the pH of three samples, while a final sample was not pHadjusted. The three pH adjusted CS powder samples were individuallyadjusted to a pH of 7.0, a pH of 4.5, and a pH 0.5. The pH of the slurryincreases gradually due to the reaction between calcium silicate andHCl. Accordingly, HCl solution was added continuously to keep the pH ofthe slurry constant. Following acid treatment for 40 min at theirrespective pH values, the powders were washed with deionized water anddried at 100° C. over night. The powders discussed herein and preparedaccording to this exemplary process at an exemplary slurry pH of 7.0,4.5, and 0.5 are referred to as CS-7.0, CS-4.5, and CS-0.5,respectively. Non-acid treated calcium silicate slurry is referred to asCS.

The following example describes methods for characterization of calciumsilicate before and after acid treatment (e.g., as prepared in Examples1 and 2 described above) and results of such characterization are shownin FIGS. 2-6C.

EXAMPLE 3

Characterization of Mesoporous Calcium Silicate

The morphologies of acid-treated calcium silicate particles wereobserved using a scanning electron microscope (FESEM; FEI, SIRION, OR).The chemical compositions of calcium silicate particles before and afteracid modification were examined by energy-dispersive X-ray spectroscopy(EDS). An X-ray diffractometer (XRD; PW 3040/00 X'pert MPD, Philips,Eindhoven, the Netherlands) was used to analyze the phase composition ofcalcium silicate particles. Infrared spectra were recorded by a Fouriertransform infrared spectrometer (FTIR, Nicolet 6700, ThermoFisher,Madison, Wis.). Composition structure analysis of calcium silicatebefore and after acid modification was demonstrated by a ²⁹Simagic-angle spinning (MAS) nuclear magnetic resonance (NMR) study. MASNMR was performed on a Varian Inova 500 (11.75 T) spectrometer operatingwith a magnetic filed of 79.5 MHz and a spinning rate of 4 kHz. A 7 mmzirconia rotor was used for all experiments. Cross-polarization (fromproton) spectra were collected for 10,000 scans with a recycle intervalof 6 seconds. Chemical shifts are referenced using an external standardof tetramethylsilane (TMS).

FIG. 2 is a XRD spectra of calcium silicate before (CS) and after acidmodification (CS-7, CS-4.5, CS-0.5) in accordance with an embodiment ofthe disclosure. As shown in FIG. 2, samples having acid modificationshow an amorphous hump between the 20° and 30° regions, which can beattributed to the presence of hydrated silica. This hump was moreobvious when calcium silicate was treated at a lower pH value (e.g., pHof 0.5). Referring to FIG. 2, the spectrum of CS-0.5 was similar to thatof silicic acid (H₂SiO₃).

Table 1 shows the results of EDS analysis of calcium silicate (CS)before and after acid modification. As shown in the second column, Cacontent decreased significantly following acid modification.

TABLE 1 EDS analysis of Calcium Silicate before and after acidmodification. Material Ca (at %) Si (at %) O (at %) CS 18.83 23.05 58.12CS-7 7.36 27.20 65.44 CS-4.5 1.37 28.81 69.83 CS-0.5 — 28.44 71.56

The leaching of Ca²⁺ from CS results in the formation of a modifiedsurface layer which is calcium depleted and silicon enriched. Whenmodified at a lower pH, more calcium silicate can react with HCl. Forexample, when treated at pH 0.5, mesoporous CS transformed completelyinto hydrated silica, and no calcium was detected by EDS, as shown intable 1.

FIG. 3 is a FTIR spectra of calcium silicate before and after acidmodification in accordance with an embodiment of the disclosure. Asshown in FIG. 3, CS showed strong adsorption at 1050 and 800 cm⁻¹, whichcan be attributed to the Si—O—Si stretching vibration. The band near 450cm⁻¹ corresponds to the Si—O bond rocking [36]. The significantdifference between the particles before and after modification appearedat 950 cm⁻¹ and can be interpreted as Si—OH bond stretching. The band at1630 cm⁻¹, which arose from the O—H bending, was also found aftermodification. The bump in the 3500-3300 cm⁻¹ regions, due to O—Hstretching and adsorbed water, was apparent in the acid-modifiedparticle spectra [36]. As demonstrated and shown in FIG. 3, bandsrepresenting Si—OH and O—H increase with a decrease of pH value.

The chemical shift of ²⁹Si is affected by the silicon coordinationnumber and by its first neighbors (bridging and nonbridging oxygen). TheQ^(n) species (where n=0-4 is the number of bridging oxygen molecules)all exhibit different chemical shifts, and can thus theoretically beseparated by NMR [42]. FIG. 4 is a ²⁹Si magic-angle spinning (MAS)nuclear magnetic resonance (NMR) analysis of calcium silicate before(CS) and after (CS-7) acid modification in accordance with an embodimentof the disclosure. Referring to the MAS NMR results shown in FIG. 4,non-acid modified calcium silicate (CS) demonstrates a major peak Q²near −85 ppm. The major peak Q² has previously been demonstrated forsilicate chains [37]. Following acid modification at pH 7.0, the Q² peakis minimally detectable, while hydroxylated (OH) Q³ (at −102 ppm) and Q⁴(at −110 ppm) peaks are observed. Peaks Q³ and Q⁴ have previously beendemonstrated for silica gel [37]. The results indicate the formation ofsilica gel reactive layer with OH group after acid modification, whichis consistent with the results from the XRD and FTIR analyses.

FIGS. 5A-5C are, respectively, scanning electron microscopy (SEM) imagesof morphologies and microstructures of calcium silicate following acidtreatment at (A) pH 7.0, (B) pH 4.5 and (C) pH 0.5, and in accordancewith an embodiment of the disclosure. The reactive layer on the surfaceof calcium silicate following acid modification showed porousnanostructures. The size of these newly formed nanoparticles on sampleCS-7 was between 40 and 60 nm. Following treatment at pH 0.5, thenanoparticle size was further decreased to between 20 and 30 nm.

To calculate pore size, the CS powder samples were characterized bynitrogen gas adsorption/desorption isotherms at 77 K and measured usinga surface analyzer (TRISTAR 3000, Micromeritics, Norcross, Ga.). In thisexample, samples were pretreated by heating at approximately 300° C. for4 hours to remove physically adsorbed gases and/or moisture from thesample surfaces. Pore size distributions were calculated from thedesorption branch of the isotherm using the Barrett-Joyner-Halenda (BJH)method, and surface areas were measured using the Brunauer-Emmett-Teller(BET) method.

FIGS. 6A-6C are, respectively, graphical representation of a pore sizedistribution for mesoporous calcium silicate samples CS-7, CS-4.5 andCS-0.5, and in accordance with an embodiment of the disclosure. FIG. 6Ashows CS-7 having a wide pore size distribution; however, a sharp peakdetected at around 4 nm suggests a mesoporous reactive layer formationat the particle surface. FIGS. 6B and 6C show CS-4.5 and CS-0.5,respectively, having a narrow pore size distribution when compared tothe pore size distribution observed for CS-7 samples. For example, theaverage pore diameter was 5.0 nm for CS-4.5 and 4.4 nm for CS-0.5.

Table 2 shows the BET specific average surface areas for mesoporouscalcium silicate samples before and following acid modification.

TABLE 2 Properties of Mesoporous Calcium Silicate BET surface area Porevolume Average pore diameter Material (m²/g) (cm³/g) (nm) CS 65.5 CS-7221.3 0.575 9.7 CS-4.5 332.9 0.475 5.0 CS-0.5 356.2 0.582 4.4

The BET surface area of non-acid modified CS was 65.5 m²/g. In contrast,mesoporous calcium silicate samples having acid modification demonstratean increase in BET surface area. For example, following acid treatmentof calcium silicate at pH 7, 4.5 or 0.5, BET surface areas were 221.3m²/g, 332.9 m²/g and 356.2 m²/g, respectively.

XRD analysis of acid modified calcium silicate under differentconditions showed the formation of an amorphous silicon-enriched layer,which was similar to silicic acid (FIG. 2). In FIG. 3, FTIR furtherdemonstrated this reactive layer contained high amounts of O—H and Si—OH(silanols) groups. These results indicate that a hydrated silica gellayer having abundant Si—OH bioactive groups was formed on the surfaceof calcium silicate following acid modification.

Furthermore, acid modification creates a mesoporous surface layer oncalcium silicate. FESEM microstructure analysis showed that followingacid modification, the newly formed silica gel reactive layer hasnanostructures, with particle sizes between 20 and 30 nm after treatmentat pH 0.5 (FIG. 5C). Nitrogen gas adsorption studies showed the size ofthese pores was about 4-5 nm. These mesoporous structures increase thesurface area on the calcium silicate particles, which can absorb higherlevels of protein and other bioactive agents.

EXAMPLE 4 Protein Adsorption by Mesoporous Calcium Silicate

Protein adsorption by mesoporous calcium silicate samples wasquantified. In this example, estimation of the amount of proteinadsorbed by mesoporous calcium silicate was determined using bovineserum albumin (BSA, A7030, Sigma, Saint Louis, Mo.) and lysozyme fromchicken egg white (L6876, Sigma, Saint Louis, Mo.) as model proteins.Some of the physical properties of BSA and lysozyme are listed in Table3 [35].

TABLE 3 Physical Properties of Model Proteins Molecular mass IsoelectricDimension Protein (Da) point (nm) BSA 66,400 4.7 4 × 4 × 14  Lysozyme14,400 11 3 × 3 × 4.5

In this example, 50 mg of non-acid modified and acid modified calciumsilicate particles (e.g., powder), prepared according to Example 2above, were immersed in 20 milliliters of a 1 mg/ml solution of eitherBSA or lysozyme at 37° C. The methods described herein were carried outat selected pH values with phosphate buffer solution (pH=7.0) and aceticbuffer solution (pH=4.5). At selected time points, the slurries werecentrifuged and the amounts of proteins in the supernatants weremeasured by Micro BCA™ protein assay kit (Pierce, Rockford, Ill.). Theprotein amount loaded on CS and mesoporous calcium silicate wascalculated by determining the change in protein concentration insolution. Precipitates in the centrifuged solution were filtered, washedwith distilled water and dried for approximately 48 hours at roomtemperature in open air. Changes in pore structure and surface chemistryof the particles following protein adsorption were determined throughnitrogen adsorption and FTIR, using the methods described above.

FIGS. 7A-7B are graphical representations of the kinetics of BSAadsorption by non-acid modified calcium silicate (CS) and by mesoporouscalcium silicate (mesoporous CS) in accordance with an embodiment of thedisclosure. FIG. 7A shows that CS and mesoporous CS samples immersed ina solution maintained at a pH of 7.0 exhibit low BSA adsorption. Incontrast, FIG. 7B shows that mesoporous CS samples immersed in asolution maintained at a pH of 4.5 exhibit an increase in BSA adsorptioncompared to samples immersed at pH 7.0 and to CS samples. As shown inFIGS. 7A and 7B, the equilibrium capacity for BSA adsorption at pH 4.5by CS-0.5, CS-4.5 and CS-7 particles was 100, 93 and 82 μg/mg,respectively.

FIG. 8 is a graphical representation of the kinetics of lysozymeadsorption by non-acid modified calcium silicate (CS) and by mesoporouscalcium silicate (mesoporous CS) at a pH of 7.0 in accordance with anembodiment of the disclosure. In comparison to the kinetics of BSAabsorption at pH 7.0 shown in FIG. 7A, the adsorption kinetics oflysozyme by calcium silicate is higher and adsorption by mesoporous CSis significantly higher. As shown in FIG. 8, the equilibrium capacitywas 195, 186 and 157 μg/mg of wollastonite for CS-0.5, CS-4.5 and CS-7,respectively. As shown in FIG. 8, the equilibrium capacity for lysozymeadsorption by CS-0.5, CS-4.5 and CS-7 all demonstrate significantlyhigher loading than the 43 μg/mg equilibrium capacity for CS.

In this example, mesoporous CS demonstrated significantly higher proteinloading (e.g., higher levels of protein adsorption) than unmodified CS.The higher protein loading capacity observed for mesoporous CS can beattributed to the formation of Si—OH functional groups in the mesoporoussurface layer.

Changes in surface chemistry of mesoporous CS particles followingabsorption of lysozyme were analyzed by FTIR. FIG. 9 shows the FTIRspectra of mesoporous calcium silicate before and following lysozymeadsorption in accordance with an embodiment of the disclosure. The amideI band near 1650 cm⁻¹ is attributed to the C═O stretching mode, and theamide II band near 1520 cm⁻¹ is attributed to the bending and thestretching mode of N—H and C—N vibrations [35]. Following lysozymeprotein loading on the mesoporous calcium silicate samples, the amide Iand amide II bands can be clearly identified. Additionally, FIG. 9 showsthat the OH⁻ band at 1630 cm⁻¹ and Si—OH band at 950 cm⁻¹ (seen in theFTIR spectra of CS-0.5 without lysozyme) decreased significantlyfollowing lysozyme protein adsorption (seen in the FTIR spectra ofCS-0.5+lysozyme), indicating interactions between protein molecules andSi—OH groups.

Retaining structural conformation and bioactivity of loaded proteins canbe important for protein delivery. Amide I and amide II bands aretypically used for indication of lysozyme structure integrity. The amideI band is due to the α-helical conformation of lysozyme, and the amideII band can be attributed to the parallel β-sheet structure of lysozyme[35]. Therefore, the appearance of amide I and amide II bands indicatesthat the structural conformation of lysozyme is retained and does notdenature after adsorption on mesoporous calcium silicate.

Changes in pore structure of mesoporous CS particles followingabsorption of lysozyme were determined by nitrogen absorption. FIG. 10is a graphical representation of nitrogen adsorption isotherms of CS-7before and following lysozyme adsorption in accordance with anembodiment of the disclosure. As shown in FIG. 10, the amount ofnitrogen absorbed by the mesoporous CS particles decreased followinglysozyme adsorption. Surface area of mesoporous calcium silicatefollowing lysozyme adsorption was calculated using the BET method asdescribed above. The BET specific average surface areas and pore volumesof mesoporous CS samples CS-7, CS-4.5 and CS-0.5 are shown in Table 4.

TABLE 4 Properties of Mesoporous Calcium Silicate following LysozymeAdsorption BET surface Pore volume Average pore diameter area (m²/g)(cm³/g) (nm)   CS-7 + lysozyme 90.5 0.26 9.2 CS-4.5 + lysozyme 142.6 0.34.4 CS-0.5 + lysozyme 139.4 0.39 4.1

Comparing the results in Tables 1 and 4, both the BET specific averagesurface areas as well as the pore volumes of all mesoporous CS sampleswere reduced following lysozyme adsorption.

Comparison of FIGS. 7A and 8 show that mesoporous calcium silicate has ahigher adsorption capacity for lysozyme that BSA. The dimension of BSA,which is about 4×4×14 nm3, is much larger than lysozyme, which hasdimensions measuring 3×3×4.5 nm3 [35]. Accordingly, lysozyme maydemonstrate more efficient loading into the nanoscale pores of themesoporous CS particles than BSA due to its smaller size. For example,the large reduction in the BET specific average surface area and porevolume (Table 4) can be attributed to the tight packing of lysozymemolecules into the pores of mesoporous CS. These results suggest thatfor mesoporous structures, the size of a protein molecule (or otherbioactive molecule) may affect the efficiency of its adsorption.

Electrostatic interactions between protein molecules (or other bioactivemolecules) and adsorbent materials can be a factor for efficientloading/adsorption. At pH 7.0, mesoporous CS particles are negativelycharged. BSA, which has an isoelectric point of 4.7, is also negativelycharged at a pH 7.0. Therefore, the repulsive interaction between BSAprotein molecules and mesoporous CS particles restricts the amount ofBSA adsorbed. In contrast, the surface charge of BSA has a negligiblerepulsive interaction with mesoporous CS particles treated at pH 4.5which can result in the remarkable increase of BSA adsorption capacity(FIG. 6).

In addition to electrostatic interaction, the Si—OH groups formed on thesurface of wollastonite particles also play a role in proteinadsorption. Protein molecules contain hydroxy groups and amino groups,which can form hydrogen bonding with Si—OH. FTIR analysis (shown in FIG.3), showed a decrease in the Si—OH (950 cm⁻¹) band after proteinadsorption, suggesting an interaction between the protein molecules andthe Si—OH groups. Due to the presence of abundant O—H and Si—OH groupsin acid modified particles, acid modified wollastonite displays higherprotein adsorption capacity than unmodified wollastonite.

Mesoporous structure with high surface area is also suggested to bebeneficial to protein adsorption [35]. The porous structure resultingfrom acid modification has small pore sizes (e.g., about 4-5 nm). Thesesmall pores are not accessible for BSA adsorption due to the largerdimensions of BSA, which is about 4×4×14 nm³. Lysozyme, which is asmaller protein with dimensions of 3×3×4.5 nm³, can be absorbed into themesopores of wollastonite. This can be a reason for higher adsorptioncapacity achieved for lysozyme compared to BSA. Lysozyme adsorption intothe mesopores can be confirmed by nitrogen adsorption isotherms ofmesoporous wollastonite before and after protein loading. The largereduction in the special surface area and pore volume can be attributedto the tight packing of lysozyme molecules in the pores of mesoporouswollastonite.

EXAMPLE 5 Protein Release from Mesoporous Calcium Silicate

In this example, lysozyme protein-loaded (e.g., approximately 50 mg)mesoporous calcium silicate particles were prepared as described inExample 4 above, and the samples were immersed in about 20 millilitersof acetic buffer solution (pH=4.5) or in about 20 milliliters ofphosphate buffer solution (pH=7.0). At selected time points, slurrieswere centrifuged and released protein in the supernatant was measured byMicro BCA™ protein assay kit.

FIGS. 11A-11B are graphical representations showing the cumulativerelease kinetics of lysozyme from protein-loaded calcium silicate andmesoporous calcium silicate samples in accordance with an embodiment ofthe disclosure. As shown in FIG. 11A, the release rate of lysozymeprotein from CS at a solution pH of 7.0 was very rapid. For example, aninitial burst of released protein (e.g., about 28%) was detected in thesupernatant within the first 6 hours. The protein concentration of thesupernatant remained nearly constant over the next 7 days, indicating nofurther or little release of protein from the CS particles following theinitial burst. In contrast to the release kinetics demonstrated bylysozyme protein loaded CS, Figure 11A shows differing protein releasekinetics for the lysozyme protein loaded mesoporous CS particles. Asshown, the release kinetics displayed a two-step release patternincluding an initial small burst release followed by a relatively slowsustained release over the following 7 days. The initial burst in thefirst 6 hours for CS-7, CS-4.5 and CS-0.5 samples was around 16.7%,14.1% and 13.5%, respectively. Although the rate at which the proteinwas released decreased during the next 7 days, gradual protein releasefrom the mesoporous CS samples was still detectable. After 7 days, thecumulative amount of released protein for CS-7, CS-4.5 and CS-0.5 was26.8%, 22.2% and 20.8%, respectively.

FIG. 11B shows the release rate of lysozyme protein from CS andmesoporous CS samples. As shown, the release rates of all samples at asolution pH of 4.5 demonstrated a remarkable increase over the releaserates of the same samples at a solution pH of 7.0 (FIG. 11A). At pH 4.5,the cumulative 7 day lysozyme protein release for CS-7, CS-4.5 andCS-0.5 was 68%, 59% and 54%, respectively.

EXAMPLE 6 Degradation of Mesoporous Calcium Silicate

In this example, estimation of the degradation rate of mesoporouscalcium silicate of Example 2, was determined. 50 mg of mesoporous CSparticles was immersed in 20 ml of acetic buffer solution (pH=4.5) or in20 ml phosphate buffer solution (pH=7.0). At selected time points,mesoporous CS particles were centrifuged and dried at 100° C. overnight.The degradation rate was calculated by weight change of the mesoporousCS particles before and following immersion.

FIG. 12 is a graphical representation of the degradation rate ofmesoporous calcium silicate in accordance with an embodiment of thedisclosure. As shown, mesoporous calcium silicate exhibits a highdegradation rate over 7 days. For samples immersed in phosphate buffersolution at a pH of 7.0, the amount of degraded mesoporous CS at day 7for CS-7, CS-4.5 and CS-0.5 was around 13.7%, 18.8% and 22.5%,respectively. Additionally, the degradation rate of mesoporous CSsamples immersed in acetic buffer solution at a pH of 4.5 was remarkablyincreased compared to samples incubated at a pH of 7.0.

As described above in Example 5, mesoporous calcium silicate particlesdisplay a two-step release pattern including a small initial burstrelease followed by a relatively slow sustained release, (FIGS.11A-11B). The initial burst release can be attributed the release ofprotein molecules adsorbed on an exterior surface of the mesoporous CSparticles. The entrapped proteins in the mesoporous structures can bereleased slowly as the support structure (the mesoporous CS material)degrades. Accordingly, the mesoporous structures can provide sustainedrelease kinetics over a longer period of time. Calcium silicate showshigher degradation rates when immersed in solutions at pH 4.5 ascompared to solutions at pH 7.0 (FIG. 12). These degradation ratessupport faster release of protein at a lower pH when compared to a highpH.

EXAMPLE 7 Biocompatibility of Mesoporous Calcium Silicate

Calcium silicate (CS) has been regarded as a candidate for bonereplacement biomaterial due to its good bioactivity properties. In vitroand in vivo studies showed that CS ceramic could induce a bone-likeapatite layer formation in simulated body fluid (SBF) [27] andchemically integrate into the structure of living bone tissue [28]. Toimprove the mechanical properties, CS coating on titanium alloy was alsodeveloped by plasma spray [29, 30]. Additionally, nano-sized calciumsilicate particles were synthesized and its biocompatibility wasdemonstrated by an in vitro study [31, 32]. The analyses of these invitro and in vivo studies reveal that the primary reason forbiocompatibility of calcium silicate is the formation of Si—OH on itssurface when exposed to body fluid.

In this example, biocompatibility of calcium silicate and mesoporouscalcium silicate particles (e.g., powder) with human fetal osteoblastcells was evaluated in vitro. CS and mesoporous CS samples (e.g., CS-7,CS-4.5, CS-0.5) were sterilized by autoclaving at 121° C. for 30minutes. An established human fetal osteoblast cell line, hFOB 1.19(ATCC, Manassas, Va.), was used in this study. The cells were seeded in24-well plates with 5 g of calcium silicate (CS or mesoporous CS)particles per well. Initial cell density was about 2.0×10⁴ cells perwell. The base medium used for this cell line was a 1:1 mixture of Ham'sF12 Medium and Dulbecco's Modified Eagle's Medium (DMEM/F12, Sigma, St.Louis, Mo.), having 2.5 mM L-glutamine (without phenol red). The mediumwas supplemented with 10% fetal bovine serum (HyClone, Logan, Utah) and0.3 mg/ml G418 (Sigma, St. Louis, Mo.). Cultures were maintained at 34°C. under an atmosphere of 5% CO₂. Medium was changed every 2-3 days forthe duration of the experiment.

Cell proliferation was evaluated using an MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assayfollowing 1, 4 and 7 days of incubation. An MTT (Sigma, St. Louis, Mo.)solution of 5 mg/ml was prepared by dissolving MTT in PBS, followed byfilter sterilization using a 0.2 micron pore filter paper. The MTTsolution was diluted 1:9 (50 μl into 450 μl) in DMEM/F12 medium, and 500μl of diluted MTT solution was then added to each sample in the 24-wellplates. After 2 hours of incubation, 500 μl of solubilization solutionmade up of 10% Triton X-100, 0.1N HCl and isopropanol were added to thewells to dissolve the formazan crystals. 100 μl of each resultingsupernatant was transferred into a 96-well plate, and read by a platereader at 570 nm. Data are presented as mean±standard deviation.Statistical analysis was performed using Student's t-test, and P<0.05was considered statistically significant. CS samples were used ascontrols for evaluating cell proliferation of hFOB cells incubated withmesoporous CS samples.

FIG. 13 is a graphical representation of cell densities of human fetalosteoblast cells cultured with samples of CS and mesoporous CS inaccordance with an embodiment of the disclosure. As shown, cellproliferation was evident for hFOB cells in culture with CS andmesoporous CS samples following 1, 4 and 7 days. As shown in FIG. 13,for each CS or mesoporous CS sample, cell density increased with anincrease in culture time. Statistical analysis showed that cell densityon mesoporous CS samples was significantly higher than those onunmodified CS samples.

To observe cell-material interaction, calcium silicate and mesoporouscalcium silicate powders were pressed by uniaxial pressing with apressure of 10 MPa, and sintered at 1100° C. for 2 hours. Human fetalosteoblast cells (hFOB cells) were seeded onto the pressed samples withthe in vitro seeding process as described above. After culturing for 3and 7 days, samples were fixed with 2% paraformaldehyde/2% osmiumtetroxide (OsO4) for 2 hours at room temperature. The fixed samples werethen dehydrated in an ethanol series (30%, 50%, 70% 95% and 100% threetimes), followed by a hexamethyldisilane (HMDS) drying procedure. Aftergold coating, the samples were observed under SEM for cell morphologies.

FIGS. 14A-14B are SEM images of morphologies of cells cultured onpressed mesoporous calcium silicate (CS-7) after 3 days (14A) and 7 days(14B) of culture in accordance with an embodiment of the disclosure. Asshown in FIG. 14A, cells exhibited a typical osteoblast phenotype withsome lamellipodia and filopodia extensions, indicating good cellattachment and spreading on the mesoporous CS-7 surface. After 7 days ofculture, a confluent layer of cells were observed on the CS-7 materialsurface (FIG. 14B).

The osteoblast cell culture test shows that CS has goodbiocompatibility, and hFOB cells attach and proliferate on CS. Comparedto unmodified CS particles, mesoporous CS powders display higherbiocompatibility. Unmodified CS may undergo a reaction in cell culturemedium resulting in a decrease in pH of the media. This pH decrease mayattenuate cell growth. The mesoporous CS samples have been shown to bestable in the cell culture medium. Another important reason for highbiocompatibility of mesoporous CS may be attributed to the active OHgroups on the acid-modified CS surface.

C. REFERENCES

The following references are herein incorporated by reference.

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D. CONCLUSION

Mesoporous calcium silicate (e.g., wollastonite) was prepared by acidmodification of calcium silicate particles, through the formation of ahydrated silica gel layer having Si—OH functional groups on the surfaceof the calcium silicate particles. This surface layer had a mesoporousstructure having pore diameters around 5 nm (e.g., below 10 nm).Mesoporous calcium silicate particles showed BET specific averagesurface area as high as 356 m²/g following acid modification at pH 0.5.Protein adsorption studies indicated that mesoporous calcium silicatehad a higher capacity for BSA and lysozyme adsorption than unmodifiedcalcium silicate. The release kinetics, discussed above, show thatproteins on mesoporous calcium silicate can be released continually overone week, whereas the proteins on unmodified particles showed burstrelease within a few hours. Methods disclosed herein can be used forsynthesis of mesoporous wollastonite that can be used as a carrier forbioactive agents, such as proteins and/or drugs, and for theircontrolled release during bone regeneration and other biomedicalapplications.

From the foregoing, it will be appreciated that specific embodiments ofthe disclosure have been described herein for purposes of illustration,but that various modifications may be made without deviating from thescope of the disclosure. Aspects of the disclosure described in thecontext of particular embodiments may be combined or eliminated in otherembodiments. While features and characteristics associated with certainembodiments of the disclosure have been described in the context ofthose embodiments, other embodiments may also exhibit such features andcharacteristics, and not all embodiments need necessarily exhibit suchfeatures and characteristics to fall within the scope of the disclosure.The following examples reflect further embodiments of the disclosure.

1. A mesoporous calcium silicate composition for controlling release ofa bioactive agent, comprising: acid-modified calcium silicate particles,the calcium silicate particles having a mesoporous structure; and abioactive agent.
 2. The composition of claim 1 wherein the mesoporousstructure includes exposed Si—OH function groups, and wherein thebioactive agent is hydrogen bonded to the mesoporous structure.
 3. Thecomposition of claim 1 wherein the mesoporous structure includes ahydrated silica gel layer formed on a surface of the mesoporousstructure.
 4. The composition of claim 1 wherein the bioactive agentincludes a protein.
 5. The composition of claim 1 wherein the bioactiveagent includes at least one of a growth factor, a pharmaceutical agent,and a vitamin.
 6. The composition of claim 1 wherein the bioactive agentincludes an osteoporotic drug.
 7. The composition of claim 1 wherein arelease profile of the bioactive agent includes a burst phase and anextended release phase.
 8. The composition of claim 7 wherein theextended release phase is a slow release phase extending for a pluralityof days.
 9. The composition of claim 7 wherein: the acid-modifiedcalcium silicate particles includes a first portion of acid-modifiedcalcium silicate particles having a first bioactive agent adsorbedthereon, the first bioactive agent having a first release profile; andthe composition further includes a second portion of acid-modifiedcalcium silicate particles having a second bioactive agent adsorbedthereon, the second bioactive agent having a second release differentfrom the first release profile.
 10. The composition of claim 1 whereinthe bioactive agent is a first bioactive agent, and wherein thecomposition includes a second bioactive agent.
 11. The composition ofclaim 1 wherein the composition is biocompatible and bioresorbable, andwherein biocompatible comprises biocompatibility with respect to atleast one of eukaryotic cells, mammalian cells, bone forming cells,osteoblast cells, cartilage cells, muscle cells, stem cells,differentiated stem cells, bone marrow stem cells and nerve cells. 12.The composition of claim 1 wherein the composition is used forregenerating tissue, and wherein the tissue includes at least one ofbone, cartilage, muscle and musculoskeletal tissue.
 13. The compositionof claim 1 wherein the acid-modified calcium silicate particles includesat least one dopant, and wherein the dopant includes one or more of ametal salt with metal ions and a metal oxide.
 14. A biocompatiblecomposition for controlling release of a bioactive agent, comprising:mesoporous calcium silicate particles; a bioactive agent, wherein thebioactive agent is adsorbed on the particles; and a calcium-basedceramic.
 15. The composition of claim 14 wherein the calcium-basedceramic includes at least one of calcium phosphates and calciumsulfates.
 16. The composition of claim 14 wherein at least one of themesoporous calcium silicate and the calcium-based ceramic includes atleast one dopant, and wherein the dopant includes one or more of a metalsalt with metal ions and a metal oxide.
 17. The composition of claim 16wherein the dopant can include one or more of Zn²⁺, Mg²⁺, Si²⁺, Na⁺, K⁺,Sr²⁺, Cu²⁺, Fe³⁺/Fe²⁺, Ag⁺, Ti⁴⁺, CO₃ ²⁻, F⁻, MgO, ZnO, NaF, KF,FeO/Fe₂O₃, SrO, CuO, SiO₂, TiO₂, Ag₂O and CaCO₃, present in an amountbetween 0 and about 10 wt %.
 18. The composition of claim 14 wherein thebioactive agent is a first bioactive agent, and wherein the compositionincludes a second bioactive agent adsorbed on the particles.
 19. Amethod for synthesizing a mesoporous calcium silicate composition forcontrolling release of a bioactive agent, comprising: preparing acalcium silicate slurry from calcium silicate particles; and treatingthe calcium silicate slurry with an acid to produce a mesoporousstructure on the calcium silicate particles to form the mesoporouscalcium silicate composition, whereby the mesoporous calcium silicatecomposition controls release of a bioactive agent.
 20. The method ofclaim 19 wherein treating the calcium silicate slurry with an acidincludes treating the calcium silicate slurry with a hydrochloric acidsolution.
 21. The method of claim 19 wherein treating the precipitateslurry with an acid includes adding an acid to adjust a slurry pH toapproximately pH 0.5 to approximately pH 7.0.
 22. The method of claim 19wherein the acid is added continuously to maintain a constant slurry pH.23. The method of claim 19, further comprising adsorbing a bioactiveagent by the mesoporous calcium silicate composition from a bioactiveagent solution.
 24. The method of claim 23 wherein the bioactive agentsolution includes two or more types of bioactive agents, and whereinadsorbing a bioactive agent by the mesoporous calcium silicatecomposition includes adsorbing the two or more types of bioactiveagents.
 25. The method of claim 23 wherein the mesoporous calciumsilicate composition is a first mesoporous calcium silicate compositionand the bioactive agent is a first bioactive agent, and wherein themethod further comprises combining the first mesoporous calcium silicatecomposition having the first bioactive agent with a second mesoporouscalcium silicate composition having a second bioactive agent, whereinthe second bioactive agent is different the first bioactive agent. 26.The method of claim 23 wherein adsorbing a bioactive agent includesadsorbing a protein molecule.
 27. The method of claim 23 whereinadsorbing a bioactive agent includes adsorbing at least one of a drug, agrowth factor, a vitamin, and a morphogen.
 28. The method of claim 19,further comprising placing the mesoporous calcium silicate compositionunder body, body fluid or simulated body fluid conditions.
 29. Themethod of claim 19, further comprising precipitating calcium silicateparticles from a mixture of Ca(NO₃)₂.4H₂O and Na₂SiO₃ 9H₂O.
 30. Themethod of claim 19 wherein the mesoporous structure includes poreshaving a pore size of approximately 4 nm to approximately 10 nm.
 31. Abone graft material for musculoskeletal tissue regeneration, comprising:a mesoporous calcium silicate composition including a bioactive agentadsorbed therein, the bioactive agent having an extended release profilefrom a mesoporous structure on a surface of the composition when placedunder body fluid conditions.